Our lab has developed Replication Initiation Point (RIP) mapping and identified the start site of continuous DNA synthesis (TP = transition Point) on the top strand of DNA in locus II/9A of the fly, Sciara coprophila. TP1 is adjacent to the Origin Recognition Complex (ORC) binding site thus extending the yeast and viral paradigm that the start site for DNA sysnthesis is adjacent to the binding site of the initiator protein. Is there a specific DNA sequence recognized as an origin in metazoa? To address this in Sciara, I will complete RIP mapping of locus II/9A, extending preliminary data mapping TP2 on the bottom strand, several hundred bp from TP1 on some DNA molecules and at TP2 on others. I will use emetine to block discontinuous (Okazaki fragment) synthesis prior to RIP mapping to identify all TPs at this locus. Next, I will examine whether ORC binds adjacent to TP2. Mutations will test if a 14 nt sequence present at both TPs is a consensus sequence necessary for ORC binding. Finally, I will extend these findings to locus II/2B in Sciara. Both loci are sites where controls that normally allow an origin to be activated only once per cell cycle are overridden at a specific developmental stage, resulting in DNA amplification. These experiments will be the foundation to identify cis-acting elements for re-replication control.